Method of cell detection

ABSTRACT

A method of distinguishing between living and dead cells in a sample, the method includes: (a) contacting the sample with a viability probe which modifies the nucleic acid of dead cells within the sample; and (b) detecting nucleic acid from the cells in the sample, wherein detection involves binding of one or more oligonucleotide probes to a target sequence within the nucleic acid. A method of detecting cells in a sample, the method includes: (a) contacting the sample with a viability probe which labels the nucleic acid of dead cells within the sample; (b) separating the nucleic acid from the cells into labelled and non-labelled fraction; and (c) detecting the nucleic acid in one or both of the fractions.

FIELD OF THE INVENTION

The present invention relates to methods of assaying cells in abiological sample, in particular to methods which can distinguishbetween living and dead cells.

DESCRIPTION OF THE RELATED ART

In many circumstances it is desirable to investigate a population ofcells within a sample in such a way that qualitative or quantitationinformation relating to living cells only within that population isobtained or a comparison between the total population of living and deadcells and living cells only can be made. This is particularly the casewhen monitoring a patient for vital or bacterial infection or thepresence of cancerous cells and assessing the efficacy of a treatmentregimen therefor or for analysis of an environmental sample that may becontaminated, e.g. a water sample.

Quantification of viable bacterial populations is important in manyareas of microbiology, including public health, biotechnology, the food,water and pharmaceutical industries and in environmental studies. Asdiscussed by Sheridan, G. E. C. et al. in Applied and EnvironmentalMicrobiology, April 1998 pp. 1313-1318, conventional methods fordetecting bacterial pathogens typically involve culturing the organismsin selective media and identifying isolates according to theirmorphological, biochemical and/or immunological characteristics. Thesemethods take several days to perform and in any event, identificationmethods based on phenotypic properties are rather unreliable. Moreover,bacteria may be in a state where they are viable and infectious but notculturable and thus not detectable by these traditional methods.

Flow cytometry can be used to perform dead cell discrimination based onthe dramatic increase in cell membrane permeability observed on celldeath. The techniques may involve physical separation methods or bebased on differential light scatter or staining by fluorescent probes.In these cases, discrimination takes place at the whole cell level.

However, methods of detection and identification of cell populationswhich rely on direct nucleic acid assays, e.g. which involve anoligonucleotide probe, are capable of being very specific and can alsobe extremely sensitive, when coupled, for example, with PCR. However, asstated by Sheridan et al. supra, a significant disadvantage of DNA-basedmethods is that they cannot distinguish between living and deadorganisms. This is because DNA is rather stable and survives intactalthough the organism is dead and is therefore able for example to actas a template for PCR. Nucleic acid-based methods which could be applieddirectly to samples to give an indication of the viability of certaincell populations, e.g. microbes, would be of great significance for manyindustrial, environmental and medical applications.

Attempts have been made to devise assays which rely on detection of RNAwhich is generally much less stable than DNA and therefore could be anindicator of the number of live cells in a sample. However the resultsof these experiments suggest that RNA may not in fact yield accurateinformation as to whether the cells are alive or dead. The stability ofthe RNA molecule is sequence dependent and may vary by several orders ofmagnitude. Furthermore, both the stability and expression levels areaffected by environmental conditions (McKillip, J. L. et al., Appl.Environ. Microbiol. [1998] 64 pp 4264-4268 and Norton, D. M. et al.,Appl. Environ. Microbiol. [1999] 65 pp 2122-7).

Thus a need remains for a detection method which can utilise thespecificity and sensitivity of a nucleic acid based assay while allowingdiscrimination between live and dead cells within the cell population ofinterest.

SUMMARY OF THE INVENTION

The inventors have found that certain viability probes can be used whichselectively interact with nucleic acid from dead cells and thus allowdiscrimination between dead and live cells at the nucleic acid level,typically by influencing the generation of a signal in a nucleic-acidbased assay. The dead-cell nucleic acid is effectively exposed due tothe permeability of the cell membrane in dead cells while the nucleicacid in live cells is protected and remains unmodified by the viabilityprobe.

Thus, according to one aspect of the present invention is provided amethod of detecting cells in a sample, said method comprising:

(a) contacting the sample with a viability probe which modifies thenucleic acid of dead cells within the sample; and

(b) detecting nucleic acid from the cells in the sample.

Generally, the method of the invention will be performed so that onlyliving cells within the sample are actually detected. The method of theinvention allows differentiation between those cells in the sample whichare dead and those which are living; therefore, alternatively viewed,the present invention provides a method of distinguishing between livingand dead cells in a sample, said method comprising:

(a) contacting the sample with a viability probe which modifies thenucleic acid of dead cells within the sample; and

(b) detecting nucleic acid from the cells in the sample.

As discussed in more detail below, the ‘modification’ will typically beby way of labelling the nucleic acid and/or inactivating it. Not all thenucleic acid within all the dead cells will necessarily be modifiedduring the method. Equally, some of the living cell nucleic acid may bemodified. A viability probe may be chosen which is selective for aparticular population of cells within the sample. The viability probemay also be selective amongst the nucleic acid within a given cell, forexample modifying DNA in preference to RNA. The nucleic acid detectionmethod will typically be designed to target sequences of nucleic acidwhich would have been significantly modified in dead cells by theparticular viability probe used. The nucleic acid may be inactivated inthat its ability to generate a signal according to the detection meanschosen is prevented or significantly reduced.

Inter alia, the method of the present invention provides a simple methodfor the separate detection, using a nucleic acid-based assay, of livingand dead bacteria. The method can be applied under natural conditions toanalyse complex communities.

Alternatively viewed, the invention provides a method of excluding deadcells from an analysis of a cell population within a sample, said methodcomprising:

(a) contacting the sample containing said cell population with aviability probe which modifies the nucleic acid of the dead cells withinsaid cell population; and

(b) detecting nucleic acid from the cell population.

The term ‘cell population’ refers to the cell types which the method isbeing performed to detect. This may be all the cells present in thesample, all the prokaryotic cells present in the sample or any othergroup of cells which it is desired to investigate. The cell populationwill be any distinguishable class or group of cells which can beidentified by the nucleic acid detection method. Thus, a particulardetection method will be chosen on the basis of how specific the cellpopulation of interest is. Therefore regions within the nucleic acidwhich will be targets for the detection method will be common to all thecells within the cell population of interest. The target region may becommon to all prokaryotic cells or just a particular strain ofmicroorganism, depending on the specificity of the assay method and thecell types/microorganisms which it is desired to detect.

By “viability probe” is meant any agent which is able to enter deadcells but not living cells. It will be appreciated that a suitable agentneed not be 100% selective, i.e. it is possible that small amounts ofthe agent will be able to enter living cells. Preferably if the amountof the viability probe is present in a typical dead cell, i.e. theaverage concentration of the agent in a dead cell is assigned a value100, then the amount present in a typical living cell, i.e. the averageconcentration in a living cell will be less than 30, preferably lessthan 20, more preferably less than 10, e.g. less than 5.

Suitable viability probes are well known in the art and documented inthe literature. According to the present invention, the viability probemust be able to interact with nucleic acid in the dead dells, bybinding, e.g. covalently or through hydrogen bonding, or in any otherway. As discussed in more detail below, the viability probe willtypically act by inactivating or labelling the dead cell-originatingnucleic acid.

Suitable agents may be chemicals such as dyes which associate withnucleic acid or enzymes, for example DNAses or RNases or restrictionenzymes. Non-enzymatic viability probes e.g. chemicals are preferred asthey are smaller and better able to cross the dead cell membranes,although still unable to breach living cell membranes.

Peptides and oligonucleotides, e.g. antisense sequences or PNA probesare further examples of suitable viability probes. These are generallysmall and therefore can enter dead cells more easily than enzymes. Thesemolecules may be able to prevent or reduce nucleic acid detectiondirectly, e.g. by inhibiting PCR but more usually they will be used tolabel the dead cell nucleic acid in a way which can then be utilised toseparate dead cell nucleic acid from nucleic acid derived from livingcells. Alternatively, a moiety may be bound to thepeptide/oligonucleotide which can modify the nucleic acid to inhibitsignal generation, e.g. after photo-induced covalent binding. Thepeptide/oligonucleotide is thus acting as a carrier and may allow forspecific binding and modification of a targeted region of nucleic acid.Suitable inhibiting moieties include EMA and 4′-AMDMIP.

Dyes which act as living/dead markers are known in the art and includeethidium bromide monoazide (EMA), 4′-AMDMIP, AMIP,5-MIP(5-methylisopsoralen) (Cimino, G. D. et. al. Nucleic Acids Research(1991) Vol. 19. No. 1 pp 99-107), SYTO 59 red fluorescent nucleic acidstain and SYTOX green nucleic acid stain, LIVE/DEAD BacLight BacterialViability Kit, ViaGram Red+Bacterial Gram Stain and Viability Kit(Molecular Probes). Further agents include BODIPY FL-14-dUTP, fluorescinconcanavalin A, propidium iodide, YO-PRO-1 dye, Alexa Fluor 488 annexinV, C4-BODIPV 500/510 CY and Hoechst 33258 (Molecular Probes) which areparticularly suitable for use in methods which relate to assays ofeukaryotic cells wherein some of the cells have undergone apoptosis.

Many suitable viability probes are fluorescent dyes but it is not theability to fluoresce which is primarily being utilised in order todistinguish between living and dead cells according to the methods ofthe present invention. Instead, it is the ability of these agents toassociate with and in some way alter the character of the nucleic acidfrom dead cells which enables differentiation. The viability probe mayremain bound to the nucleic acid (i.e. the dead-cell originating nucleicacid is labelled) and this association between the viability probe andthe nucleic acid utilised to separate dead cell originating nucleic acidfrom live cell originating nucleic acid.

This is preferably achieved by providing a binding partner to theviability probe, typically attached to a solid support. For example, anantibody to the viability probe may be provided on a solid support andthe nucleic acid from the sample contacted with the solid support toallow specific binding of the viability probe/dead cell nucleic acidcomplex to the antibody attached to the solid support. The living celloriginating nucleic acid with no associated viability probe would notbind and can simply be run off and assayed. This method has theadvantage that the nucleic acid which is attached to the solid supportvia the viability probe and the antibody thereto could be eluted andassayed separately; in this way information regarding the size of thedead cell population within the sample can be generated.

Thus, according to one embodiment of the present invention is-provided amethod of detecting cells in a sample, said method comprising:

(a) contacting the sample with a viability probe which labels thenucleic acid of dead cells within the sample;

(b) separating the nucleic acid from the cells into labelled andnon-labelled fractions; and

(c) detecting the nucleic acid in one or both of the fractions.

The present invention also provides a method of differentiating betweenliving and dead cells in a sample, said method comprising:

(a) contacting the sample with a viability probe which labels thenucleic acid of the dead cells;

(b) separating the nucleic acid from the cells into labelled andnon-labelled fractions; and

(c) detecting the nucleic acid in one or both of the fractions.

Chemicals such as the dyes discussed above can act in this way, as canoligonucleotides, peptides, polypeptides, proteins and fragmentsthereof.

However, in a preferred embodiment the viability probe inactivates thedead-cell nucleic acid and thus may conveniently be referred to as an“inactivator”. By “inactivates” it is meant that the viability probeprevents or greatly reduces any signal being generated by the nucleicacid during the nucleic acid detection step. This could be achieved bydegradation of the dead cell nucleic acid using DNases or RNases orcutting specific regions using restriction enzymes. Chemicals can beused as viability probes to inactivate the nucleic acid, by degradationor by an alternative modification which in some way inhibits the nucleicacid assay, i.e. prevents the dead-cell nucleic acid acting as atemplate in the detection step. Typical of such an inactivator is EMAwhich is capable of binding covalently to DNA upon photoactivation andis able to inhibit PCR when covalently bound to or otherwise associatedwith DNA. 4′-AMDMIDP has also been shown to inhibit PCR by covalentattachment to DNA (Cimino, G. D. et al., supra).

A number of different techniques for detecting nucleic acids are knownand described in the literature and any of these may be used accordingto the present invention. At its simplest the nucleic acid may bedetected by hybridisation to a probe and very many such hybridisationprotocols have been described (see eg. Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.). Most commonly, the detection will involve an insitu hybridisation step, and/or an in vitro amplification step using anyof the methods described in the literature for this. Thus, as mentioned,techniques such as LAR, 3SR and the Q-beta-replicase system may be used.However, PCR and its various modifications eg. the use of nestedprimers, will generally be the method of choice (see eg. Abramson andMyers, 1993, Current Opinion in Biotechnology, 4: 41-47 for a review ofnucleic acid amplification technologies).

Other detection methods may be based on a sequencing approach, forexample, the minisequencing approach as described by Syvänen andSöderlund, 1990, Genomics, 8: 684-692.

The results of the PCR or other detection step may be detected orvisualised by many means, which are described in the art. For examplethe PCR or other amplification products may be run on an electrophoresisgel eg. an ethidium bromide stained agarose gel using known techniques.Alternatively, the DIANA system may be used, which is a modification ofthe nested primer technique. In the DIANA (Detection of ImmobilisedAmplified Nucleic Acids) system (see Wahlberg et al., Mol. Cell Probes4: 285(1990)), the inner, second pair of primers carry, respectively,means for immobilisation to permit capture of amplified DNA, and a labelor means for attachment of a label to permit recognition. This providesthe dual advantages of a reduced background signal, and a rapid and easymeans for detection of the amplified DNA.

The amplified nucleic acid may also be detected, or the resultconfirmed, by sequencing, using any of the many different sequencingtechnologies which are now available, eg. standard sequencing, solidphase sequencing, cyclic sequencing, automatic sequencing andminisequencing.

The step of nucleic acid detection will preferably involve binding ofone or more oligonucleotide probes (conveniently referred to herein as adetection probe) to a target nucleic acid sequence. These probes may beprimers for a nucleic acid amplification reaction, such as PCR, RT-PCR,NASBA, isothermal amplification, ligase chain-reaction etc.Alternatively, a labelled probe may be used and assayed directly.Preferably, the nucleic acid detection step will involve anamplification reaction. The use of detection probes means that targetnucleic acid sequences can be selected which are specific to the cellpopulation of interest, e.g. to a particular species or strain ofmicroorganism.

Clearly, where the nucleic acid has been degraded, e.g. by DNases orRNases or cut in a relevant region by restriction enzymes, the detectionprobes will be unable to bind to the target nucleic acid sequences andso the dead cell originating nucleic acid is unable to act as a templateand generate a signal. All the signal generated, e.g. followingamplification and separation by gel electrophoresis, will thereforederive from the nucleic acid of living cells. In this way theinformation about the living cell population in a sample is generated.Such information, as discussed above, is often more valuable thanknowing the size of a population of cells which includes living and deadcells.

Inactivators which modify the nucleic acid in other ways may alsoprevent signal generation via the viability probes. Binding of theinactivator to the nucleic acid may physically prevent binding of thedetection probe or the operation of enzymes involved in the detectionmethod, e.g. a polymerase. Enzymes such as polymerases may be inhibitedby the viability probe; it has been shown, for example, that EMA caninhibit PCR even when it is not covalently bound to the nucleic acid.The viability probe may remain associated with the nucleic acid, actingas an inactivator or label (e.g. covalently bound EMA).

Alternatively, the viability probe may modify the nucleic acid toinactivate and/or label it but will not itself remain associatedtherewith (e.g. enzymes or chemicals which degrade the nucleic acid).Methyltransferases (McClelland, M. et al., Nucleic Acids Research (1998)22, 17, pp 3640-3659) may label dead cell nucleic acid. The modificationcaused by the viability probe will preferably be non-reversible, e.g. asa result of covalent bonding to the dead cell nucleic acid ordegradation thereof. Viability probes which themselves label the nucleicacid will preferably associate strongly with the nucleic acid.Modifications caused by viability probes which do not themselves remainassociated with the nucleic acid will preferably be essentiallynon-reversible, at least under the conditions of the detection method.

The viability probe will preferably bind to the nucleic acid, thisbinding may be temporary, as in the case of a DNase. Binding includescovalent binding as well as by hydrogen bonding or any other form ofassociation.

Typically, after contacting the sample with a viability probe which isable to selectively enter dead cells and thus modify the nucleic acidtherein, the cells are lysed and the nucleic acid from the whole sampleisolated for performance of the detection method. Thus, in a preferredembodiment, the nucleic acid from all the cells in the sample is mixedprior to the nucleic acid detection method. Information about just thelive cell population or in certain circumstances just the dead cellpopulation can be obtained because of the physical difference betweenlive and dead cell nucleic acid which has been caused by the viabilityprobe. As discussed above, where the viability probe is a DNase orRNase, the ‘difference’ will be that the dead cell nucleic acid does notexist as such, as it has been degraded. Typically, the viability probeenzyme e.g. DNase, is denatured on cell lysis and so is not available todegrade the nucleic acid from living cells.

The present methods can therefore be contrasted with those ofdifferentiation by flow cytometry, which rely on a discrimination at thecellular level which generally depends on a visual assessment of whetherthe whole cell is dead or living. Thus there will be a sorting orphysical separation of the living and dead cells during flow cytometry.The fact that the sample can be contacted with a viability probe andthen a nucleic acid detection step performed on the sample without everhaving to differentiate living from dead cells makes the method of thepresent invention very convenient. In addition, the method can be fullyautomated and does not require any judgement to be made, such as whengating cells during flow cytometry, as to whether any given cell isliving or dead. Also, only liquid samples with low particle amounts canbe processed by flow cytometry.

As mentioned above, a nucleic acid detection method which involves anamplification step is preferred. Amplification primers are added to thesample and the well known cycles of the selected amplification protocolperformed. The dead cell originating nucleic acid may have beenenzymatically degraded and thus only the live-cell originating nucleicacid can be amplified. Alternatively the dead-cell nucleic acid may havebeen chemically modified so that the amplification reaction isinhibited, possibly by preventing primer binding or enzyme function. Inany event, the amplification product will be derived solely oressentially from the living-cell nucleic acid and can be analysed byconventional techniques to obtain an estimate of the amount of targetnucleic acid in the original sample and thus of the number of livingcells within a given cell population in the sample. Conveniently, theamplification products will be stained, e.g. using ethidium bromide andthen separated, e.g. on an agarose gel.

The methods of the invention may conveniently be performed utilising theprotocol described in WO98/51693. Thus, the cells in the sample arecaptured by binding to a solid support, the cells are contacted with theviability probe and then the cells are lysed. The released nucleic acidis then bound to the same solid support before being analysed. Asdiscussed in WO98/51693, the contents of which are incorporated hereinby reference, the bound nucleic acid may be analysed while still boundto the solid support or eluted from the solid support for performance ofthe detection method. The solid support will preferably comprise aparticulate material, preferably magnetic beads. Cells and nucleic acidare conveniently bound to the sold support by precipitation.

In a particularly preferred embodiment, bacteria from e.g. a foodsample, are captured on a solid support, the bacteria are then contactedwith the viability probe e.g. EMA and then exposed to light. DNA is thenpurified and analysed. The use of a magnetic or magnetisable solidsupport enables convenient manipulation of the cells/nucleic acid duringthe method.

The term “cell” is used herein to include all prokaryotic (includingarchaebacteria) and eukaryotic cells and other viable entities such asviruses and mycoplasms. All cells must show a significant increase inthe ability of certain agents to cross the outer membrane, capsid etc.on cell death. Representative “cells” thus include all types ofmammalian and non-mammalian cells, plant cells (provided the selectedviability probe is able to cross the cellulose cell wall), algae, fungi,protoplasts, bacteria, bacteriophages, mycoplasms, protozoa and viruses.

The sample may thus be any material containing nucleic acid within suchcells, including for example foods and allied products, clinical andenvironmental samples. Thus, the sample may be a biological sample whichmay contain viral or other cellular material as discussed above.Representative samples thus include whole blood and blood-derivedproducts such as plasma or buffy coats, urine, faces, cerebrospinalfluid or any other body fluids, tissues, cell cultures, cell suspensionsetc., and particularly environmental samples such as soil, water or foodsamples.

The sample may also include selectively pure or partially purifiedstarting material, such as semipure preparations obtained by a cellseparation process.

The difference between a living cell and a dead cell is not easy todefine in general terms for all cells but for the purposes of thepresent invention, distinction relies on the fact that a dead cellexhibits greatly enhanced permeability to viability probes. Generally,live cells will be culturable, i.e. can multiply to form colonies onagar plates or produce visible turbidity in broth. Such a definition maynot be appropriate in all circumstances, e.g. in the case of viruseswhich require host cells to replicate. Nevertheless, for a given targetcell of interest, parameters can readily be established as indicative ofa living cell, crucial to the determination will be the ability of aviability probe to enter the cell. In the case of bacteria, it should benoted that bacteria in a viable but non-culturable state (i.e. which canbe ‘resuscitated’ to the normal culturable state under certainconditions) are considered to be living and their membranes do not haveenhanced permeability.

The nucleic acid which is modified by the viability probe may be DNA,RNA or any naturally occurring or synthetic modification thereof, andcombinations thereof. Preferably however the nucleic acid will be DNA,which may be single or double stranded or in any other form, e.g. linearor circular.

The viability probe may modify the nucleic acid in an indiscriminatemanner, e.g. following binding by or in a discriminate manner targetedto a particular region of the nucleic acid e.g. by use of a restrictionendonuclease. Generally there will be no discrimination, other thanbetween living and dead cells, at the viability probe level. Theviability probe will typically be able to enter different types of deadcells of which the cell population of interest is only a proportion.Alternatively, viability probes may be selected which are able to enterthe dead cells of interest more easily than other dead cells i.e. theviability probe is able to target the cells of interest. This enables alower overall concentration of viability probe to be used and mayenhance the selectivity of the method.

The nucleic acid detection can introduce a level of specificity whichcan distinguish between different populations of living cells. Forexample, a probe can be used which binds to a target region of nucleicacid which is particular to a certain microorganism which it is desiredto assay. Likewise eukaryotic cells of interest can be assayed byselection of a probe which hybridises to a target region of nucleic acidwhich is specific to a certain cell type, for example to a particularcancer cell. As discussed above, such probes may be labelled orconveniently be used in an amplification reaction.

A further advantage of the methods of the present invention is theability of the viability probe to modify free nucleic acid present inthe sample, i.e. nucleic acid which is not contained within cells—eitherliving or dead. The modification may be by any of the methods describedabove, including enzymatic degradation, chemical modification to inhibitthe nucleic acid acting as a template in a detection method andlabelling to allow segregation of nucleic acid from living cells fromother nucleic acid in the sample. Thus free nucleic acid in the samplecan be excluded from the assay. The methods can accurately determine thepopulation of living cells within the sample and not over-exaggerate theliving cell population because of signal generated by dead cells or freenucleic acid. Thus in a preferred embodiment of the present invention,the viability probe modifies the nucleic acid of dead cells and freenucleic acid within the sample.

The method of the present invention is suitable for analysing cell deathitself, particularly by necrosis and apoptosis. Necrosis typically beinginduced by extracellular conditions and resulting in an early membranepermeabilization; apoptosis being associated with programmed cell deathand exhibiting a rather delayed permeability in the cell membrane(O'Brien, M. and Bolton, W., Cytometry (1995) 19, pp 243-255). Thenumber of dead cells can be analysed by comparing the results of amethod of the present invention as compared to a method which does notdistinguish between living and dead cells and uses no viability probe.Alternatively, when the present invention is performed in such a waythat the modification caused by the viability probe is used to separatethe dead cell population from the living cell population, thenqualitative and quantitative information about the dead cell populationcan be obtained directly.

The various reactants and components required to perform the methods ofthe invention may conveniently be supplied in kit form. Such kitsrepresent a further aspect of the invention.

At its simplest, this aspect of the invention provides a kit fordetecting cells in a sample comprising:

(a) a viability probe;

(b) means for performing nucleic acid detection.

The invention thus also provides a kit for distinguishing between livingand dead cells in a sample comprising:

(a) a viability probe;

(b) means for performing nucleic acid detection.

Means (a) and (b) will be as described and discussed above, in relationto the methods of the invention. (b) will typically comprise one or moredetection probes as defined herein. The kit will optionally contain (c),means for lysing the cells. In those methods wherein the living and deadcell nucleic acid fractions are separated prior to nucleic aciddetection, the kit may optionally contain (d), a binding partner to theviability probe which is preferably bound to a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the drawings in which:

FIG. 1 shows the results on EtBr-stained agarose gel electrophoresis ofthe separation of PCR amplification products of DNA which had beencontacted with EMA. pos=sample without EMA; neg=sample without DNA;mw=lambda BsteII cut molecular weight marker.

FIG. 2 shows the results of-EtBr-stained agarose gel electrophoresis ofthe separation of PCR amplification products of DNA from living bacteriatreated with EMA (A), and signal intensities for the EMA treated samplesrelative to the untreated samples (B) (L)=unexposed and living;(E)=ethanol; (H)=heat and (F)=formamide treatment. (+) EMA added; (−)=noEMA added.

Panel A: The ethidium bromide stained (10 μg/ml) amplification productswere separated on a 1% agarose gel for 45 minutes at 100 volts.neg=sample without DNA; mw=lambda BsteII cut molecular weight marker.

Panel B: The signal intensities were determined as pixel values in an 8bit grayscale by using the program Gel-Pro ANALYZER software (MedicaCybernetics, Silver Spring. Md.). The relative signal intensities forthe EMA treated samples are given as a percentage of the untreatedsamples.

FIG. 3 provides graphical representation of the effect of externallyadded DNase on the stability of DNA in heat-treated cells. Cells wereincubated at (A) 20° C., (B) 55° C., (C) 72° C., (D) 100° C., (E) 121°C., for 5 min (A-D) or 15 min (E) before DNase was added. DNA wasquantified by 5′-nuclease PCR after 5, 15 and 30 min, 1, 6 and 24 hrs inboth DNase-treated and negative controls. The stability of purified DNAtreated with DNase was investigated (F). The amount of DNA with (♦), andwithout (□) DNase treatment, are given relative to the amount before theheat-treatment. The error bars are standard deviations.

DETAILED DESCRIPTION EXAMPLE 1

PCR Inhibitor Effect of EMA Covalently Bound to DNA ThroughPhotoactivation

The PCR inhibitory effects of both photoactivated, and unactivated EMAwere investigated for pure DNA. PCR amplifications were carried out ondilution series of 1000, 100 and 10 μg/ml EMA. The 16S rDNA gene wasamplified through the application of the forward primer5′TGGCTCAGATTGAACGCTGGCGGC3′ and the reverse primer5′CTTGTTACGACTTCACCCCA3′. These primer sites are universally conservedfor eubacterial 16S rDNA.

The PCR amplifications were done using the GeneAmp 9700 PCRamplification system (PE biosystems, Norwalk, Conn., USA). The 50 μl PCRreactions contained 10 pmol of each primer, 200 μM dNTP, 10 mM Tris-HCl(pH 8.8), 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton® x-100, 1 U DynaZyme™Thermostable DNA Polymerase (Finnzymes OY, Espoo, Finland) and 2 ngtemplate. The cycling parameters were: 94° C. for 30 s, 55° C. for 30 sand 72° C. for 1 minute.

The cycling step was repeated 25 times. The samples were heated to 94°C. for 5 minutes prior to the amplification, and an extension step at72° C. for 7 minutes was included after completion of the amplificationreactions.

For each dilution series PCR was employed both on samples where EMA hadbeen covalently photoactivated to DNA through illumination with a Luxor18W type FL-185 lamp (Luxor ASA, Oslo, Norway) at 5 cm for 15 minutes,and on samples where EMA had been added to the DNA in the dark. For thehighest concentration (1000 μg ml), both the photoactivated and nonactivated EMA inhibited the PCR reaction. For 100 and 10 μg/ml, thephotoactivated EMA inhibited PCR, while PCR for the unactivated EMA wasnot inhibited (FIG. 1). 10 μg/ml amplification products were separatedon a 1% agarose gel for 45 minutes at 100 volts. The conclusion fromthese experiments is that EMA covalently bound to DNA is sufficient toinhibit PCR amplification, and that EMA by itself is only inhibitory toPCR at high concentrations.

EXAMPLE 2

Application of EMA for Selective PCR Amplification of DNA from Living E.coli

E. coli was killed by ethanol, heat, and formaldehyde to test the effectof EMA as a living/dead stain. In these experiments aliquots ofapproximately 10⁸ cells of E. coli were pelleted in a microcentrifuge at8000 rpm for 5 minutes, the supernant removed, and the cells resuspendedin 50 μl of the respective liquids; water 96% ethanol, and 2%formaldehyde. The ethanol and formaldehyde treated samples wereincubated at room temperature, while the water samples were incubatedboth at room temperature and at 95° C. All samples were incubated at therespective temperatures for 5 minutes.

The bacteria were subsequently pelleted by centrifugation, thesupernatant removed, and the cells resuspended in 50 μl water. EMA wasadded at a concentration of 10 μg/ml to the test samples, while for thecontrols, no EMA was added.

All the samples were exposed to light for 10 minutes. Subsequently, thesamples were resuspended in 500 μl 4 guanidine thiocyanate (GTC) and 1%sarkosyl containing 1U Dynabeads DNA DIRECT beads (Dynal AS, Oslo,Norway). Incubation was continued at room temperature for an additional5 minutes. 1 ml 96% ethanol was added, and after 5 minutes the sampleswere washed twice with 1 ml 70% ethanol. The bead and DNA complex wasthen resuspended in 50 μl water, and finally heated to 65° C. for 10minutes to remove residual ethanol. 2% of the purified DNA was used ineach amplification reaction as described above.

There were significant differences in the PCR signal intensities betweenthe controls and the EMA sample for all the killing means, while therewere no differences for the living bacteria (FIG. 2). The difference insignal intensities between the treated sample and the control were about100% both for the bacteria killed with ethanol and the bacteria killedby heat, while the difference was 85% for the formaldehyde treatedsample. The differences in effects may possibly be related to how thekilling agents permeabelize the cell membranes. Alcohols and heat act bydestroying cell membranes, while formaldehyde is an alcylating agentwith no profound effect on cell membranes. There did not seem to be adifference in the effect of EMA related to whether the DNA is single ordouble stranded. The PCR inhibition was 100% both for the ethanol(double-stranded DNA), and the heat (single-stranded DNA) killedbacteria. Increasing the amount of EMA to 100 μg/ml did not result in abetter discrimination between the viable and dead bacteria. However,this EMA concentration reduced the amplification efficiency for theliving bacteria.

Comparison of signal intensities for the controls indicated that theyield of the DNA purification and/or amplification efficiency isaffected by the treatment schemes. Both the heat-treated, andformaldehyde treated samples gave a lower yield as compared to theuntreated samples and the samples treated with 96% ethanol.

EXAMPLE 3

Effect of DNase on Heat-treated Campylobacter jejuni Type Strain DSMZ4688^(T) (Deutsche Samlong von Mikroorganismen und Zellkulturn GmbH,Braunshweig, Germany

Campylobacter jejuni is recognised as a leading human food-bornepathogen. The traditional diagnostic testing for C. jejuni is notreliable due to special growth requirements, and the possibility thatthis bacterial can enter a viable but non culturable state (VNC). Thefollowing experiment describes a 5′-nuclease PCR assay for quantitativedetection of C. jejuni, wherein external DNases are added to reduce thePCR signal from free DNA and the exposed DNA of dead bacteria.

The C. jejuni strain was plated on blood agar; 1.5 ml from 48 hr culturewas pelleted at 6000×g for 7 min at 4° C., washed and resuspended inwater, and transferred to microcentrifuge tubes.

The effect on the DNA stability at room temperature after heat-treatmentof the cultures, and after addition of DNase was investigated.1.7±0.8×10⁷ cells (with DNase) and 5.0±0.6×10⁷ cells (without DNase) ofC. jejuni type strain were used in these experiments. The cultures wereincubated at 20, 55, 72 and 100° C. for 5 min, and 121° C. for 5 min.One set of tubes were then incubated further at room temperature, andsamples for PCR analysis removed at intervals of 5, 15, and 30 min, 1 h,6 and 24 hrs, and 5 days. 10 U RQl DNase (Promega, Madison, Wis., USA)and 1× DNase buffer were added to another set of tubes before theincubation at room temperature. Aliquots were analyzed after 5, 15 and30 min, and after 1 hr, 6 and 24 hours. For the PCR analysis, DNA waspurified from 10 μl aliquots from the respective time-points, and thesubsequent 5′-nuclease PCR assay performed as described below.

DNA Isolation

Dynabeads® DNA Direct I (Dynal AS, Oslo, Norway), 200 μl, were thenadded to the suspension of bacteria, and the bacteria-bead suspensionwas incubated at 65° C. for 20 minutes, followed by incubation at roomtemperature for another 2 minutes. DNA bound to magnetic beads was thendrawn to the wall of the microcentrifuge tube by a magnet (MPC®-E, DynalAS, Oslo, Norway) for 2 minutes. The supernatant containing salts,detergent and cell debris was carefully removed without disrupting theDynabeads/DNA complex. The beads were washed twice with a washing buffer(buffer 2 from the kit). Finally, the DNA was removed from the beads byresuspending in 40 μl 10 mM Tris HCl, pH 8.0 (buffer 3 from the kit),and incubation at 65° C. for 5 minutes. The beads, now released from theDNA, were collected with the magnet, and the DNA-containing supernatantwas transferred to a fresh tube and used directly in the PCR.

TaqMan Probe and Primer Design

The probe regions were localized in the completed C. jejuni strain NCTC11168 genome sequence (http://www.sanger.ac.uk/Projects/C. jejuni.). ThePrimer Express™ (version 1.0), ABI Prism™ (PE Biosystems, Foster City,Calif., USA) was used for the primer-probe design, together withguidelines from PE Biosystems. The GCG version of FastA was used tosearch for sequence similarities to other known sequences.

PCR Fragment Specificity

Specific PCR primers and probe were designed for C. Jejuni. The proberegion was chosen to optimize specificity and amplification efficiency.First, putative probes were constructed using the primer expressprogram, and then these probes were subjected to a FastA search(Pearson, W. R. and D. J. Lipman, 1991, Improved tools for biologicalsequence comparison, Proc. Natl. Acad. Sci. USA 85: 2444-2448) in theEMBL database (release 60). An 86 bp fragment located in position 381121to 381205 relative to the published C. jejuni strain NCTC 11168 genomesequence (http://www.sanger.ac.uk.Projects/C. Jejuni/) was identified inthese screenings. There were no known sequences in the EMBL databasewith significant homology to this probe region. The most closely relatedsequence had 57.7% identity, and was located in the putative gene YonOin the complete sequence of Bacillus subtilis strain 168. PCR primerswere constructed from the positions 381121-381144 (forward), and381205-381185 (reverse), while the probe is located in the position381146-381181 (Table 1).

After the probe region was identified on a theoretical basis, thespecificity of the selected primers and probes were subjected to aempirical screening. A total of 32 C. jejuni isolates, including thetype strain, were tested and found specific to the chosen primers andprobe. The specificity of the primers and probe were controlled against13 Campylobacter strains of 11 other species, and a set of 41 speciesbelonging to other genera of phylogenetically related or commonfoodborne organisms and pathogens, all of which were found negative. Inthese experiments, the quality of the purified DNA was verified throughamplification with universal 165 rDNA PCR primers. In addition, aqualitative PCR with the amplification primers alone was done forselected strains. These experiments confirmed that the amplificationprimers are specific for C jejuni. Unspecific PCR products were notdetected. No false negatives were recorded among the 32 isolates testedand no false positives were recorded among the other Campylobacterspecies, or strains belonging to other genera. This demonstrates thehigh specificity of the designed primer-probe set. Furthermore, theamplification primers alone were also specific for C. Jejuni avoidingpotential artefacts in mixed population due to competition for theamplification primers through amplification of targets from otherbacteria.

5′-Nuclease-based PCR Assay

Amplification reactions (50 μl) contained a DNA sample (1 μl), 1×TaqMan™ Buffer A, 5 mM MgCl₂, 200 μM dATP, dCTP, dGTP, and 400 μM dUTF,0.02 μM. C. Jejuni specific probe, 0.3 μM C. jejuni specific primers—seeTable 1 below (each), 1 U AmpErase™ Uracil N-glycosylase (UNG), and 2.5U AmpliTaq Gold™ DNA polymerase.

TABLE 1 Primers and fluorogenic probe Denaturation Probe or primerSequence (5′-3′) temp (° C.)* Primers Fwd CTG AAT TTG ATA CCT TAA GTGCAG C 60.4 Rev AGG CAC GCC TAA ACC TAT AGC T 60.3 Probe TCT CCT TGC TCATCT TTA GGA TAA ATT 66.6 CTT TCA CA *Calculated by nearest-neighboralgorithm by the Primer Express ™ program (primer concentrations 300 nM,probe concentration 20 nM, salt concentration 55 nM).

PCR samples and controls were prepared in triplicate. Reaction tubeswere MicroAmp® Optical tubes and tube caps were MicroAmp® Optical caps.All consumables were supplied by PE Biosystems, Foster City, Calif.,USA.

Before amplification, the PCR mixture was heated to 50° C. in 5 min tolet the UNG destroy possibly contaminating PCR products, and 95° C. for10 min to denature the template DNA. Amplification, profile: 40 cyclesof 95° C. for 20 seconds and 60° C. for one minute. Reactions wereperformed in the ABI Prism® 7700 Sequence Detection System (PEBiosystems, Foster City, Calif., USA). Reaction conditions wereprogrammed and data were analyzed on a power Macintosh 4400/20 (AppleComputer, Santa Clara, Calif., USA) linked directly to the ABE prism®7700 Sequence Detection system using the SDS 1.6.3 application software(PE Biosystems, Foster City, Calif., USA) as described by themanufacturer.

PCR products were detected directly by monitoring the increase influorescence from the dye-labelled C. jejuni specific DNA probe. TheTaqMan probe consisted of an oligonucleotide with a 5′-reporter dye anda 3′-quencher dye. The reporter dye, FAM (carboxyfluorescein) wascovalently linked to the 5′ end of the oligonucleotide. The fluorescenceof the reporter was quenched by TAMRA(6-carboxy-N,N,N′,N′-tetramethylrhodamine), located at the 3′ end. Whenthe probe was intact, the proximity of the reporter dye to the quencherdye resulted in suppression of the reporter fluorescence. If the probewas cleaved, the reporter and quencher dyes were separated, causing thereporter dye fluorescence to increase.

The amplification was plotted as ΔR_(n), which was the normalizedreporter signal (reporter signal minus background), against number ofcycles. A threshold signal was chosen, which intersected theamplification curves in the linear region of the semi-log plot. Thisgave threshold cycles (C_(T)), which is defined as the PCR cycle wherean increase in fluorescence first occurred, for each amplification plot.Different amplifications could then be compared by their respectivethreshold cycles. The C_(T)-values were plotted against log inputDNA/cells and gave standard curves for quantification of unknown samplesand possibilities to estimate the amplification efficiency in thereaction (Heid, C. A., J. Stevens, K. J. Livak and P. M. Williams, 1996,Real time quantitative PCR, Genome Research 6: 986-994; PE Biosystems,1997, User bulletin 2; ABI PRISM 7700 Sequence Detection System).

The PCR product was verified with ethidium bromide-stained 2% agarosegels (SeaPlaque® GTG®, Agarose, FMC BioProducts, Rockland, Me., USA).Agarose gel electrophoresis was performed essentially as described bySambrook et al. (1989) in Molecular cloning: a laboratory manual 2nd ed.Cold Spring Harbour Laboratory Press.

Effect of Externally Added DNases on the Stability of DNA inHeat-Treated Cells

The effect of externally added DNases was compared to control samples inwhich no DNase was added in cultures incubated at 20, 50, 72 and 100° C.for 5 min, and 121° C. for 15 min. Time series for incubation at roomtemperature (20° C.) after the heat treatment are shown in FIG. 3.

There was little difference between the DNase treated samples and thecontrol at 20° C. and 55° C. (FIG. 3A and B). For the samples incubatedat 55° C. the signal stabilized relatively rapidly at 1 to 3% of theinput signal (FIG. 3B). For 72, 100 and 121° C. the addition of DNaseresulted in a nearly instant 1 log reduction of the signal compared tocontrol without DNase. No further reductions in the signals wereobserved neither for the controls, nor the DNase treated samples until24 hours after the heat treatment (FIGS. 3C, D and E).

The effect of DNase treatment was also investigated on purified DNA fromC. jejuni. This DNA was very rapidly degraded, and after 30 minutes onlyabout 0.01% of the input material was left. This fraction, however,seemed stable for at least 24 hours after the treatment (FIG. 3F).

The ability of DNase to selectively degrade free DNA and DNA inheat-killed Campylobacter to further reduce the signal generated fromthe dead cells were investigated. There were no significant differencesbetween the DNase treated, and the untreated samples at 20 or 55° C. The20° C. experiments show that DNA within intact cells are not degraded byexternally added DNases, and did not result in a signal reduction.Although no viable cells could be recovered after the 55° C. treatment,this temperature did not seem sufficient to expose the DNA to externallyadded DNases. For the samples heated to 72, 100 and 121° C. the additionof DNases nearly instantly reduced the amount of template by 1 logcompared to the, untreated samples. For these temperatures the majorfraction of DNA in the killed cells was not detected by the assay. Inconclusion, applying DNase treatment to reduce the noise signalgenerated from dead bacteria seems promising for samples that have beentreated at temperatures above 72° C. are further examples of suitableviability probes for 5 min or more. This could be of particular utilitywhen organisms have been killed by pasteurization and it is desired toanalyse a population introduced after pasteurization. Other temperaturestested simulate alternative methods of treating food to killmicroorganisms, such as boiling, 100° C. and canning, 121° C.

EXAMPLE 4

A further series of experiments has been performed using real timequantitative PCR (TaqMan PCR).

TaqMan-based PCR Assay

Amplification reactions (50 μl) contained a DNA sample (1-5 μl), 1×TaqMan™ Buffer A, mM MgCl₂, 200 μM dATP, dCTP, dGTP, and 400 μM dUTP,0.1 μM E. coli specific probe, 0.3 μM E. coli specific primers (each),and 2.5 U AmpliTaq GoldT™ DNA polymerase. PCR samples and controls wereprepared in triplicates. Reaction tubes were MicroAmp® Optical tubes andtube caps were MicroAmp® Optical caps. All consumables were supplied byPE Biosystems, Foster City, Calif., USA. Before amplification, the PCRmixture was heated to 95° C. for 10 mins to denature the template DNA.Amplification profile: 40 cycles of 95° C. for 20 seconds and 60° C. forone minute. Reactions were performed in the ABI prism® 7700 SequenceDetection System (PE Biosysrems, Foster City, Calif., USA).

The amplification was plotted as ΔR_(n), which was the normalizedreporter signal (reporter signal minus background), against number ofcycles. One then chose a threshold signal, which intersected theamplification curves in the linear region of the semi-log plot. Thisgave threshold cycles (C_(T)), which is detined as the PCR cycle wherean increase in fluorescence first occurred, for each amplification plot.Different amplification could then be compared by their respectivethreshold cycles.

The light activation was done on ice with a OSRAM SLG1000 lamp (OSRAM,Norway) with a 650 W halogen light bulb at a distance of 10 cm.

Results

Time Series for Covalent Binding of EMA to DNA

EMA in concentrations 2 ng/μl, 20 ng/μl and 100 ng/μl were added topurified DNA, and exposed to light for 5 s, 15 s, 45 s, 1 min and 3 min.The amount of DNA that could serve as a template was then tested by realtime quantitative PCR. The binding reaction was complete at about 30 s,with a difference in the PCR cycles (ΔCT) until a detectable sigital wasobtained at approximately 12 for all concentrations tested—indicatingthat a fraction of ½¹²=0,000244 could serve as a PCR template aftercovalent binding of EMA to DNA.

Time to Light Inactivation of EMA

The EMA was exposed to light for 5 s, 15 s, 30 s, 45 s 1 min, 3 min and5 min prior to binding to DNA (light activation for 1 min). The EMAconcentrations 2 ng/μl, 20 ng/μl and 100 ng/μl were tested. There was nosignificant difference between the control and the test sample for EMAexposed to light for 30 s or more prior to binding to DNA. Thisindicates that the ability of EMA to inhibit PCR is rapidly destroyed bylight exposure.

This is a very useful property which means that exposure of the reactionto light after a period sufficient to allow the EMA to enter the deadcells and bind to the nucleic acid can effectively ‘freeze’ the systemat that point. This obviates any possible problems with free EMAdistorting the results.

Viable/dead Studies on E. coil 0157 Strain 765 [Matforsk, AS, Norway]

The strain was killed with 70% isopropyl ethanol for. 30 min. 10 ng/μland 100 ng/μl EMA were added both to the viable control, and to thekilled bacteria. The bacteria were exposed to EMA for 30, 1 min, 5 min,10 min and 15 min. Exposure to 100 ng/μl for 5 min give the bestdiscrimination with approximately 1 cycle difference for the viablebacteria, and 10 cycles (½¹⁰) for the killed bacteria. Results indicatedthat for untreated, i.e. viable bacteria, C_(T) was at 20 PCR cycles forbacteria not exposed to EMA and 21 cycles for bacteria exposed to EMA.For treated, i.e. dead bacteria, C_(T) was at 20 PCR cycles for bacterianot exposed to EMA but at 30 for bacteria exposed to EMA. Microscopicexamination of the staining of the viable population indicated thatthere was a fraction of dead bacteria, which can explain the slightdifference between the control and the EMA treated bacteria.

1. A method of excluding dead cells from an analysis of a cellpopulation within a sample, said method comprising: (a) contacting thesample with a viability probe which modifies the nucleic acid of deadcells within the sample, wherein said viability probe selectivelyinteracts with nucleic acid from dead cells, such that said nucleic acidin living cells remains unmodified by the viability probe; (b) lysingthe cells after step (a) and after cell lysis allowing the nucleic acidfrom both living and dead cells to mix prior to performance of step (c);and (c) detecting nucleic acid from the living cells in the sample,wherein detection involves binding of one or more oligonucleotide probesto a target sequence within said nucleic acid.
 2. A method as claimed inclaim 1 wherein according to step (c), a signal is generated from saidoligonucleotide probes which is proportional to the amount of livingcell originating nucleic acid.
 3. A method as claimed in claim 1 whereinthe majority of nucleic acid detected originates from living cells.
 4. Amethod as claimed in claim 3 wherein at least about 65% of the nucleicacid detected originates from living cells.
 5. A method as claimed inclaim 4 wherein at least 75% of the nucleic acid detected originatedfrom living cells.
 6. A method as claimed in claim 5 wherein at leastabout 85% of the nucleic acid detected originates from living cells. 7.A method as claimed in claim 1 wherein the viability probe modifies thenucleic acid by at least one of labelling the nucleic acid andinactivating the nucleic acid.
 8. A method as claimed claim 1 whereinthe viability probe selectivity modifies DNA.
 9. A method as claimed inclaim 1 wherein the detection method involves binding of anoligonucleotide probe to a target sequence within the nucleic acidmolecule from living cells.
 10. A method as claimed in claim 1 whereinthe detection method involves amplification of a region of nucleic acidfrom the living cells in the sample.
 11. A method of detecting cells ina sample, said method comprising: (a) contacting the sample with aviability probe which labels the nucleic acid of dead cells within thesample, wherein said viability probe selectively interacts with nucleicacid from dead cells, such that said nucleic acid in living cells remainunmodified by the viability probe; (b) lying the cells after step (a)and after cell lysis allowing the nucleic acid from both living and deadcells to mix prior to performance of step (c); (c) separating thenucleic acid from the cells into labeled and non-labeled fractions; and(d) detecting the nucleic acid in one or both of the fractions.
 12. Amethod as claimed in claim 1 wherein the viability probe covalentlybinds to dead cell nucleic acid.
 13. A method as claimed in claim 1wherein the viability probe is a dye, a peptide, an oligonucleotide or anucleic acid degrading enzyme, wherein said oligonucleotide comprises aninhibiting moiety, wherein said inhibiting moiety is EMA or 4′-AMDMIP.14. A method as claimed in claim 13 wherein the viability probe isselected from ethidium bromide monoazide (EMA), 4′-AMDMIP, AMIP, 5-MIP(5-methylisopsoralen) and a DNase.
 15. A method as claimed in claim 1wherein the modification caused by the viability probe isnon-reversible.
 16. A method as claimed in claim 1 wherein the viabilityprobe also modifies free nucleic acid present in the sample.
 17. Amethod as claimed in claim 11 wherein the viability probe covalentlybinds to dead cell nucleic acid.
 18. A method as claimed in claim 11wherein the viability probe is a dye, a peptide, or an oligonucleotide,wherein said oligonucleotide comprises an inhibiting moiety, whereinsaid inhibiting moiety is EMA or 4′-AMDMIP.
 19. A method as claimed inclaim 18 wherein the viability probe is selected from ethidium bromidemonoazide (EMA), 4′-AMDMIP, AMIP, and 5-MIP (5-methylisopsoralen).
 20. Amethod as claimed in claim 11 wherein the viability probe also modifiesfree nucleic acid present in the sample.